Wednesday, August 27, 2008

Journal Club: mRNA Levels are a Good Indicator of Off-targeting

Next to delivery and immune stimulation, sequence-specific off-targeting is considered one of the fundamental challenges in the development of RNAi Therapeutics. Such off-targeting largely occurs by microRNA-like gene silencing following recognition of off-target RNAs that have limited sequence complementarity to the guide strand of the siRNA. Since microRNA gene silencing had long been believed to inhibit translation without affecting mRNA levels, there has been concern that the true extent of RNAi off-targeting may be larger than originally reported in studies assessing changes in global RNA levels. By achieving the feat of simultaneously measuring microRNA-induced changes in thousands of proteins, two high-quality papers now find that, surprisingly, mRNA levels are indeed a very good indicator of off-targeting, validating the use of established high-throughput RNA assays during the siRNA selection stage of RNAi Therapeutics development.

Key to the success of the Bartel lab at MIT (Baek et al.: The impact of microRNAs on protein output) and the Rajewski lab in Berlin (Selbach et al.: Wide-spread changes in protein synthesis induced by microRNAs), was their use of the SILAC technique (Stable Isotope Labeling with Amino acids in Cell culture). In this procedure, newly synthesized proteins are labeled with amino acids containing different isotopes. To determine the impact of microRNAs on global protein synthesis (~5000 proteins were identified in the studies), the samples with and without a given small duplex microRNA were labeled with different isotopes. This results in peptides from the same protein exhibiting slightly shifted peaks during protein mass-spectrometry, and the ratio of the peak intensities belonging to a pair of peptides is taken as a measure of microRNA-mediated gene silencing (1= no change). Changes at the protein level thus determined were then compared to changes on the mRNA level by microarray analysis.

As expected, the authors found that among the down-regulated proteins were highly enriched those that contained microRNA seed target sites in their 3’ untranslated regions (UTRs). Consistent with our notion of microRNAs as regulators of a group of genes rather than individual genes, typically 200-500 proteins were changed by more than 30% following the introduction or deletion of a microRNA, with about half of them likely the consequence of a direct interaction with the microRNA. Remarkably, despite the introduction of rather large amounts of microRNAs into the tissue culture cells (25-100nM using lipofection), most of these changes were rather subtle, and very rarely were changes of more than 3-fold detected. While it is always possible that wide-spread subtle changes may lead to a directed cellular response or cause toxicity in the case of RNAi Therapeutics, this observation is in agreement with microRNAs often acting as rheostats to maintain cellular homeostasis and suggests that the introduction of random short duplex RNAs should not necessarily lead to gene regulatory chaos and distinct phenotypes.

The surprise came when it was found that changes in protein levels could for the most part be accounted by changes in mRNA levels. Only very few proteins were reduced without concomitant mRNA changes. Without going into the details, the studies nevertheless support a model in which translational repression is the primary mechanism of microRNA-mediated gene silencing and mRNA destabilization a consequence thereof. Consistent with this hypothesis,
in the rare instances where 2 microRNAs were located close to each other, silencing was synergistic, yet the mRNA level was not further reduced. This is quite interesting as it may explain how the early use of microRNA reporter systems in which multiple microRNA target sites were closely spaced and which consequently caused strong gene suppression could have led to the notion of translational silencing only. The 20-30% reduction in mRNA could have easily been dismissed as experimental noise.

For practical purposes, however, measuring mRNA levels are highly predictive of proteomic changes and this should allow for the selection of RNAi Therapeutics candidates that have an increased likelihood of not causing toxic changes in gene expression. Merck has long been a proponent of this approach and partly for this reason have closely aligned the systems biology expertise of subsidiary Rosetta Inpharmatics with their RNAi incubator Sirna Therapeutics, in addition to employing systems biology for identifying the right gene targets.

It is very comforting to know that the extent and severity of off-targeting on the protein level is not significantly greater than what we have observed at the RNA level. Moreover, the ability to use high-throughput RNA expression technologies for assessing off-targeting and chemical modification techniques which have proven to significantly further reduce off-targeting potential of siRNAs should minimize the risk of RNAi Therapeutics candidates to fail due to sequence-specific off-targeting. A good siRNA at high concentrations in tissue culture reduces about 5-10 off-target RNAs by more than 2-fold (and as we know now proteins to a similar degree, not more), while the target is the most suppressed gene. By acting at the RNA level, RNAi Therapeutics has the distinct safety advantage in that it allows for off-targeting to be assessed in such a comprehensive manner. Before investing $1 billion dollars in the development of a drug, it appears to be well worth taking advantage of this by trying to understand these changes.

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By Dirk Haussecker. All rights reserved.

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